A platform for genome mining of multidrug-resistant pathogens to develop therapeutic phages using synthetic biology

项目摘要/摘要 耐多药[MDR]病原体是全球健康威胁，也是现代医学的挑战； 而且，由于细菌对新抗生素的抗药性现在已经超过了抗生素的开发工作，因此至关重要的是 开发新的有效的抗菌剂替代品。抗菌素耐药性危机带来了新的利益 以开发基于噬菌体的疗法。在过去的十年里，美国生产噬菌体的努力 针对不同细菌病原体的治疗已显示出有希望的结果，包括成功 人类患者中危及生命的感染的治疗。噬菌体疗法的安全性仍然是一个令人担忧的问题 然而，FDA强调了对噬菌体制剂的要求：它们需要安全、纯净、有效、 完全裂解，非转导，缺乏不良基因(抗生素耐药性，毒力因子)和 细菌内毒素。 如果用于治疗的噬菌体历史上是从自然环境中分离出来的，那么最近的进展 噬菌体遗传学和基因组工程已被证明成功地产生了合成的、严格裂解的衍生物 瞄准病原体。针对MDR病原体的合成噬菌体的开发将需要管道来 加快我们对新发现的噬菌体及其合成生物学潜力的了解。批判性见解 它们的生物学特性，即基因组结构、噬菌体复制周期、遗传含量(必需基因与 可有可无的[抗生素耐药性和毒力基因])，与目标宿主的相互作用是先决条件。 在这里，我们提出了一个平台来(I)挖掘MDR病原体的基因组，这是一个识别休眠状态的金矿 溶原噬菌体直接从其自然宿主内；以及(Ii)开发高通量管道以快速 获得有关噬菌体生物学的知识，以指导我们努力设计合成噬菌体作为疗法。 我们将使用A组链球菌(GAS)，这是2019年疾控中心“18种MDR病原体”上的“令人担忧的威胁”。 观察名单，作为我们的榜样。我们证明了TN-seq能够区分功能性溶原噬菌体和隐匿性溶原噬菌体。 在GAS基因组中，以及重新启动休眠前驱体进入裂解周期的突变。在目标1中，我们将生产 指导决定选择哪种噬菌体具有治疗潜力的关键知识 生物学：我们将通过实验评估噬菌体基因组组织、噬菌体复制/转导 机制、寄主范围和细胞表面受体(S)。在目标2中，我们将实现设计-构建-测试-学习 优化合成生物学工作的“循环”流水线，即删除不需要的基因和添加 “有效载荷”基因，以增强其作为治疗噬菌体的潜力。最后，我们将使用体内创伤模型 感染，以测试我们产生的合成噬菌体的效果。我们的首要目标是开发工具 以及将我们的合成生物学噬菌体工程平台应用于其他MDR病原体的经验。

PROJECT SUMMARY/ABSTRACT Multidrug resistant [MDR] pathogens represent a global health threat and a challenge for modern medicine; and, as bacterial resistance to new antibiotics is now outpacing the antibiotic development effort, it is critical to develop new effective antimicrobial alternatives. The antimicrobial resistance crisis is bringing new interests worldwide to develop phage-based therapies. Over the last decade, efforts in the U.S. to produce phage therapeutics targeting different bacterial pathogens have shown promising results, including successful treatments of life-threatening infections in human patients. Safety of phage therapy is still a concern in the U.S.; however FDA has highlighted requirements for phage preparation: they need to be safe, pure, potent, exclusively lytic, non-transducing, and lacking undesirable genes (antibiotic resistance, virulence factors) and bacterial endotoxins. If bacteriophages for therapy have historically been isolated from natural environments, recent progresses in phage genetics and genome engineering have proven successful to generate synthetic, strictly lytic derivatives targeting pathogens. The development of synthetic phages against MDR pathogens would require pipelines to accelerate our knowledge on newly discovered phages and their potential for synthetic biology. Critical insights into their biology, i.e. genome structure, phage replication cycle, genetic content (essential genes versus dispensable [antibiotic resistance and virulence genes]), interaction with the target host, are a prerequisite. Here, we propose a platform to (i) mine the genomes of MDR pathogens, a gold mine to identify dormant lysogenic phages directly from within their natural host; and (ii) develop high-throughput pipelines to quickly gain knowledge on the phage biology to guide our efforts to engineer synthetic phages as therapeutics. We will use the Group A Streptococcus (GAS), a “Concerning Threat” on the 2019 CDC “18 MDR pathogens” Watch List, as our model. We showed that Tn-seq could identify functional lysogenic phages from cryptic ones in GAS genomes, and mutations to reboot dormant prophages into their lytic cycle. In Aim 1, we will produce the critical knowledge to guide decision on what phages to select for therapeutic potential using synthetic biology: we will experimentally assess phage genome organization, phage replication/transduction mechanisms, host range and cell surface receptor(s). In Aim 2, we will implement a design-build-test-learn cycle" pipeline to optimize the synthetic biology effort, i.e. deletion of undesirable genes and addition of “payload” genes, to enhance their potential as therapy phages. Finally, we will use in vivo model of wound infection to test the efficacy of the synthetic phages we generated. Our overarching goal is to develop the tools and experience to apply our synthetic biology phage-engineering platform to other MDR pathogens.